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Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains.
Clustered regularly interspaced short palindromic repeats (CRISPR) loci were discovered in 1987 in Escherichia coli1. However, the interest in these genetic elements increased in the early 2000s, as they were identified, along with many CRISPR-associated (Cas) proteins, in several prokaryotes2,3. Recently, it was shown that the short spacers (21-72 base pairs (bp)) between these repeats originated from extrachromosomal DNA4-7. Most importantly, it was experimentally demonstrated that those short spacers can provide resistance against bacteriophage infection and plasmid transformation8-10.
The CRISPR/Cas immune systems act in at least two general steps: (1) the adaptation stage, where new spacers derived from foreign DNA (proto-spacers) are generally acquired at the leader end of the CRISPR locus11,12; and (2) the interference stage, where the CRISPR/Cas system targets either invading DNA10 or RNA13. The mechanistic details of spacer acquisition are still unknown, but a clearer picture is emerging for the interference stage, which starts with the transcription of the CRISPR locus from a promoter located within the leader sequence14,15. The full-length RNA is subsequently cleaved by a protein or protein complex, generating shortCRISPRRNAs (crRNAs)16-19. InPyrococcus, Cas proteins use the crRNAs to target foreign RNA by complementarity in a ruler-anchoredmanner13.However, the in vivomechanismof...